CN104830750A - Method for in-vitro rapid culture of donkey skin fibroblasts - Google Patents
Method for in-vitro rapid culture of donkey skin fibroblasts Download PDFInfo
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- CN104830750A CN104830750A CN201510234370.6A CN201510234370A CN104830750A CN 104830750 A CN104830750 A CN 104830750A CN 201510234370 A CN201510234370 A CN 201510234370A CN 104830750 A CN104830750 A CN 104830750A
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Abstract
The invention relates to the technical field of culture of donkey skin fibroblasts, and particularly relates to a method for in-vitro rapid culture of donkey skin fibroblasts. The method is implemented by carrying out digestion on tissue blocks of donkey skins successively by using trypsin andcollagenase I, so that corresponding donkey skin fibroblasts are separated; and then, efficiently and quickly establishing a donkey skin fibroblast in-vitro culture system. The method disclosed by the invention can be used for solving the problem that donkey skin fibroblasts can not be cultured in vitro because donkey skins contain a large amount of collagens, and a stable donkey skin fibroblast in-vitro culture system can be established in only about three days, so that the method is short in time, and high in efficiency.
Description
Technical field
The present invention relates to donkey hide Fibroblast cell-culture technical field, specifically relate to a kind of method of donkey hide Fibroblasts in vitro fast culture.
Background technology
Donkey-hide gelatin is China's medicine, eats dual-purpose traditional Chinese medicine material, is enrich blood top grade, with ginseng, pilose antler being called " nourishing Triratna ", enjoys great prestige domestic and international market.And the skin of equine species donkey is the unique raw material producing donkey-hide gelatin.Main protein in donkey hide is I-type collagen, and polypeptides matter, polysaccharose substance and other small-molecule substances such as it and serum albumin constitute the main component of donkey-hide gelatin.But in recent years because the role of donkey as animal power is replaced by machinery gradually, add that the donkey culture-cycle is long, income is slow, China's donkey breeding stock is declined rapidly, thus makes market produce huge breach to donkey hide demand, become a large bottleneck of restriction donkey-hide gelatin industry development.
For this problem, on the one hand country has formulated corresponding strategy, encourages and expands the cultivation of donkey.On the other hand, in conjunction with the knowledge of modern biology, the utilization ratio and the new donkey-hide gelatin production ways of exploitation that improve donkey hide are also problems scientific research being badly in need of follow-up solution.First this need the problem solved to be exactly efficiently will cultivate donkey hide inoblast fast in vitro, so that research donkey hide inoblast synthesizes the Regulation Mechanism of I-type collagen further.
Cell culture processes mainly contains two kinds: tissue block method and enzyme digestion.Enzyme digestion cultivates epithelial common method, but the consumption of Digestive system, digestion temperature and time bad grasp, the results such as cell is very easily aging, apoptosis may be caused, digestion time is longer, cell is easily impaired, and terms of settlement is gradation collecting cell, thus makes experiment more loaded down with trivial details.During tissue block method's culturing cell, some tissue block only obtain less cell through long-time cultivation, and what have even acellularly climbs out of, and does not reach required cell concentration.Have no at present specially about the report that donkey hide Fibroblasts in vitro is cultivated, and the fibroblastic extracorporeal culturing method of the horse skin being all equus still adopts is traditional organize adherent method.I.e. clip one piece of skin histology, 75% alcohol-pickled about 1 minute, then rinse 3 times with containing dual anti-DPBS, to reject between skin after reticular tissue, skin histology is cut into 1cm
3the fritter of left and right, is then evenly distributed in culturing bottle, and culturing bottle adds the DMEM nutrient solution containing 10%FBS after being inverted, at 37.5 DEG C, 5%CO
2incubator in be inverted and cultivate after 4-6h, just put culturing bottle, make the slow submergence tissue block of nutrient solution, continue to cultivate.Or tissue block is directly affixed in culture dish or culturing bottle, subsequently with the DMEM nutrient solution containing 10%FBS, at 37.5 DEG C, 5%CO
2incubator in cultivate.Experiencing at least 4-6 days, or even after 18 days, the skin flbroblast of horse could be gone down to posterity, and then sets up stable cell strain, and incubation time is long, and efficiency is lower.
Summary of the invention
Technical problem to be solved by this invention is to provide a kind of method of donkey hide Fibroblasts in vitro fast culture, utilize trypsinase and Collagenase I priority digestion in the tissue block of donkey hide, corresponding donkey hide inoblast is separated, and then efficiently sets up donkey hide Fibroblasts in vitro culture system rapidly.
For solving the problems of the technologies described above, the invention provides following technical scheme: a kind of method of donkey hide Fibroblasts in vitro fast culture, comprises the following steps:
A. get one piece of donkey hide tissue, after putting into 75% alcohol disinfecting rinsing, rinse at least 3 times with the DPBS containing 100U/mL penicillin, 100U/mL Streptomycin sulphate;
B. donkey hide tissue taken out and with the knife blade after sterilizing, its epidermis and subcutaneous connective tissue rejected clean, then being cut into meat gruel shape tissue block with the surgical scissors after sterilizing;
C. meat gruel shape tissue block is put into the glass round-bottom bottle through sterilizing, add after 0.25% trypsinase hatches 1h at 37 DEG C of temperature, the Collagenase I adding 0.5% again processes 6h at 37 DEG C of temperature, visible meat gruel shape tissue block major part all disappears, can be observed suspend discrete cell and some hairs and tissue block of dissociating a little under microscope, the more fibroblastic suspension of comparatively pure donkey hide can be obtained with after 0.45 μm of metre filter;
D. by suspension centrifugal treating, supernatant discarded, add nutrient solution resuspended after proceed in culture dish, then add 1ml nutrient solution, at 37 DEG C, 5%CO
2after cultivating 30min in the incubator of concentration, will discard containing most of epithelial nutrient solution, and rejoin nutrient solution, cultivate in incubator;
E.2-3 behind sky, treat that cell covers with and get final product had digestive transfer culture, so far, stable donkey hide Fibroblasts in vitro culture system builds up.
As preferred to one of the present invention, the centrifugal treating of the suspension in described step D adopts 1000r/min, centrifugal treating 4min.
As preferred to one of the present invention, the nutrient solution in described step D comprises DMEM and 10%FBS.
As preferred to one of the present invention, the glass round-bottom bottle in described step C adopts up-small and down-big structure.
The beneficial effect that the present invention compared with prior art has is: overcome donkey hide inoblast because in donkey hide containing a large amount of collagen protein and inconvenience by a difficult problem for vitro culture, and only need within about three days, just to set up stable donkey hide Fibroblasts in vitro culture system, time is short, and efficiency is high.
Embodiment
In order to make object of the present invention, technical scheme and advantage clearly understand, below in conjunction with and embodiment, the present invention is further elaborated.Should be appreciated that specific embodiment described herein only in order to explain the present invention, be not intended to limit the present invention.
A method for donkey hide Fibroblasts in vitro fast culture, comprises the following steps:
A. with shave, the hair of a donkey small region is with it rejected clean, utilize the cutisector after sterilization to take off about 1cm
3donkey hide tissue, put into rapidly the DPBS added in advance containing 100U/mL penicillin, 100U/mL Streptomycin sulphate, take back laboratory; Taken out by donkey hide tissue with the tweezers of sterilizing, put into 75% alcohol rinsing 1min, the DPBS put into again subsequently containing 100U/mL penicillin, 100U/mL Streptomycin sulphate rinses more than three times;
B. donkey hide tissue taken out and with the knife blade after sterilizing, its epidermis and subcutaneous connective tissue rejected clean, then being cut into meat gruel shape tissue block with the surgical scissors after sterilizing;
C. meat gruel shape tissue block is put into the glass round-bottom bottle through sterilizing, wherein the selection principle of glass round-bottom bottle is adopt up-small and down-big structure, add 2ml, after 0.25% trypsinase hatches 1h in 37 DEG C of water-baths or incubator, add again 2ml, 0.5% Collagenase I at 37 DEG C of temperature, process 6h, visible meat gruel shape tissue block major part all disappears, can be observed suspend discrete cell and some hairs and tissue block of dissociating a little under microscope, the more fibroblastic suspension of comparatively pure donkey hide can be obtained with after 0.45 μm of metre filter;
D. suspension is utilized 1000r/min centrifugal treating 4min, supernatant discarded, add nutrient solution containing DMEM and 10%FBS resuspended after proceed in culture dish, then add 1ml nutrient solution, at 37 DEG C, 5%CO
2after cultivating 30min in the incubator of concentration, will discard containing most of epithelial nutrient solution, and rejoin nutrient solution, cultivate in incubator;
E.2-3 behind sky, treat that cell covers with and get final product had digestive transfer culture, so far, stable donkey hide Fibroblasts in vitro culture system builds up.
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, all any amendments done within the spirit and principles in the present invention, equivalent replacement and improvement etc., all should be included within protection scope of the present invention.
Claims (4)
1. a method for donkey hide Fibroblasts in vitro fast culture, is characterized in that, comprises the following steps:
A. get one piece of donkey hide tissue, after putting into 75% alcohol disinfecting rinsing, rinse at least 3 times with the DPBS containing 100U/mL penicillin, 100U/mL Streptomycin sulphate;
B. donkey hide tissue taken out and with the knife blade after sterilizing, its epidermis and subcutaneous connective tissue rejected clean, then being cut into meat gruel shape tissue block with the surgical scissors after sterilizing;
C. meat gruel shape tissue block is put into the glass round-bottom bottle through sterilizing, add after 0.25% trypsinase hatches 1h at 37 DEG C of temperature, the Collagenase I adding 0.5% again processes 6h at 37 DEG C of temperature, visible meat gruel shape tissue block major part all disappears, can be observed suspend discrete cell and some hairs and tissue block of dissociating a little under microscope, the more fibroblastic suspension of comparatively pure donkey hide can be obtained with after 0.45 μm of metre filter;
D. by suspension centrifugal treating, supernatant discarded, add nutrient solution resuspended after proceed in culture dish, then add 1ml nutrient solution, at 37 DEG C, 5%CO
2after cultivating 30min in the incubator of concentration, will discard containing most of epithelial nutrient solution, and rejoin nutrient solution, cultivate in incubator;
E.2-3 behind sky, treat that cell covers with and get final product had digestive transfer culture, so far, stable donkey hide Fibroblasts in vitro culture system builds up.
2. the method for donkey hide Fibroblasts in vitro fast culture according to claim 1, is characterized in that, the centrifugal treating of the suspension in described step D adopts 1000r/min, centrifugal treating 4min.
3. the method for donkey hide Fibroblasts in vitro fast culture according to claim 1, it is characterized in that, the nutrient solution in described step D comprises DMEM and 10%FBS.
4. the method for donkey hide Fibroblasts in vitro fast culture according to claim 1, it is characterized in that, the glass round-bottom bottle in described step C adopts up-small and down-big structure.
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| CN201510234370.6A CN104830750A (en) | 2015-05-05 | 2015-05-05 | Method for in-vitro rapid culture of donkey skin fibroblasts |
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| CN201510234370.6A CN104830750A (en) | 2015-05-05 | 2015-05-05 | Method for in-vitro rapid culture of donkey skin fibroblasts |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101880648A (en) * | 2010-06-25 | 2010-11-10 | 广东药学院 | Primary culture method for pancreas acinar cells |
| CN102747033A (en) * | 2012-06-26 | 2012-10-24 | 亚太干细胞科研中心有限公司 | Method for culturing mesenchymal stem cells and fibroblast tissue from gingival tissue |
| CN104087551A (en) * | 2014-07-17 | 2014-10-08 | 济南磐升生物技术有限公司 | Novel method for in-vitro separated culture of human epidermal cells |
-
2015
- 2015-05-05 CN CN201510234370.6A patent/CN104830750A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101880648A (en) * | 2010-06-25 | 2010-11-10 | 广东药学院 | Primary culture method for pancreas acinar cells |
| CN102747033A (en) * | 2012-06-26 | 2012-10-24 | 亚太干细胞科研中心有限公司 | Method for culturing mesenchymal stem cells and fibroblast tissue from gingival tissue |
| CN104087551A (en) * | 2014-07-17 | 2014-10-08 | 济南磐升生物技术有限公司 | Novel method for in-vitro separated culture of human epidermal cells |
Non-Patent Citations (3)
| Title |
|---|
| 张明 等: "大熊猫皮肤组织酶解分离效果研究", 《四川农业大学学报》 * |
| 张静南: "马、驴和骡成纤维细胞培养、核型及其G带分析研究", 《中国优秀硕士学位论文全文数据库.基础科学辑》 * |
| 杨斌 等: "组织工程皮肤种子细胞的同期快速分离", 《中国修复重建外科杂志》 * |
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